Differences In Antimicrobial Susceptibility Research Articles

Identifying LiaFSR Residues Contributing to ExPortal Integrity and Response to Antimicrobials in Group A Streptococcus

Abstract Background Group A Streptococcus (GAS), or Streptococcus pyogenes, is responsible for a myriad of diseases (pharyngitis, scarlet fever, impetigo, toxic shock syndrome) of which several are prevalent in the pediatric population. The LiaFSR two-component regulatory system (TCS) and the ExPortal functional membrane microdomain (FMM) of GAS have been shown to coordinate the response to cell membrane stress when the bacterium is exposed to antimicrobials or antimicrobial peptides (AMPs). The ExPortal coordinates translocation and processing of secreted GAS proteins and contributes to sensing membrane. In an unstressed state, LiaF acts as an inhibitor of LiaS activity, both residing within the ExPortal. In a stressed state, such as exposure to antimicrobial agents, LiaF and S dissociate, allowing LiaS to phosphorylate LiaR, resulting in subsequent activation and expression of downstream target genes. Genomic analysis indicates a strict conservation of specific protein domains and certain amino acid residues in LiaF, S, and R, which suggests their critical involvement in the LiaFSR mechanism of action. We hypothesize that conserved amino acid residues in LiaFSR, such as the LiaF C-terminal domain (CTD) and a 3xK residue motif, are essential to ExPortal formation and maintenance and may contribute to differences in antimicrobial susceptibility and virulence. Methods Two different mutant strains were analyzed and compared to (wild-type) WT: one lacking the LiaF CTD (LiaF_∆CTD) and one mutagenized in the 3xK motif (LiaF_K91-93A). Five different assays were used to examine phenotypes associated with LiaFSR activity: SpeB protease activity through bacterial growth on milk plates, ExPortal integrity via fluorescence microscopy, diamide-induced oxidative stress tolerance, antimicrobial/antimicrobial peptide (AM/AMP) susceptibility, and a direct bactericidal assay in human blood (Lancefield). For the AM/AMP susceptibility, we tested 6 different antimicrobial agents with varying mechanisms of action: bacitracin, polymyxin B, LL-37, nisin, daptomycin, and HNP-1. Results Milk plate analysis revealed a significant decrease in SpeB production in the LiaF-∆CTD vs. WT, but no difference in LiaF-K91-93A relative to WT. During fluorescence microscopy, no significant differences were detected between foci of fluorescence of either mutant strain relative to WT. When exposed to diamide-induced oxidative stress, both mutants exhibited increased tolerance relative to WT. AM/AMP susceptibility assays did not reveal any significant differences for any antimicrobial agent between either mutant strain vs. WT. Finally, the Lancefield assay revealed a significant decrease in survival in human blood between both mutant strains and WT. Mutant strains also had significantly decreased survival relative to a strain lacking LiaF (∆LiaF). Conclusions The results of the AM/AMP susceptibility assay suggest that, unlike the LiaFSR system in Enterococcus, LiaF function does not contribute to LiaFSR-dependent susceptibility. Significant differences in survival in blood indicate that the LiaF residues studied are physiologically relevant in GAS infection. However, their exact mechanism underlying this contribution is being investigated. Future research will examine downstream gene regulation to elucidate the LiaFSR-associated mechanism of decreased survival in human blood, with the aim of defining determinants of GAS bacteremia.

Antimicrobial resistance patterns and empiric antibiotic selections for patients admitted from post-acute care facilities.

To characterize the susceptibilities of positive bacterial cultures and the appropriateness of empiric antimicrobial regimens for patients admitted from post-acute care facilities (PACFs). This was a retrospective quality improvement study. The study included patients admitted from a PACF to one of 2 tertiary care teaching hospitals within the University of Pennsylvania Health System, located in Philadelphia, PA, from August 2020 to December 2021. Patients were included if they had at least one positive culture within 72 hours of admission. A total of 167 patients and 230 isolates from the study period were evaluated. The majority of positive cultures were from a urinary source (114 of 230, 49.6%). Nineteen patients (11.4%) had a history of multidrug-resistant organisms. The most common empiric antibiotics used were vancomycin (61.7%) and cefepime (59.3%). Sixty-one patients (36.5%) received inappropriate empiric therapy based on the culture results. When comparing our hospitals' general antibiogram to that of only PACF patients, Escherichia coli and Klebsiella pneumoniae had at least a 20% difference in susceptibility to levofloxacin, ceftriaxone, and cefepime. Extended-spectrum β-lactamase resistance was also higher in the PACF cohort (odds ratio, 2.09; 95% confidence interval, 1.4-3.1). Clinically significant differences in antimicrobial susceptibility were found among patients admitted from PACFs compared to our health system's general antibiogram. The increased resistance rates identified in this study support the need for hospitals to evaluate this at-risk patient population, which may drive changes to empiric antibiotic prescribing practices.

Comparative genomic analysis of Aeromonas dhakensis and Aeromonas hydrophila from diseased striped catfish fingerlings cultured in Vietnam.

Motile Aeromonas septicemia (MAS) is a burden for striped catfish (Pangasius hypophthalmus) farmers in Vietnam. MAS can be caused by several species of Aeromonas but Aeromonas hydrophila is seen as the leading cause of MAS in aquaculture, but recent reports suggest that A. dhakensis is also causing MAS. Here we investigated the bacterial etiology of MAS and compared the genomic features of A. hydrophila and A. dhakensis. We collected 86 isolates from diseased striped catfish fingerlings over 5 years from eight provinces in Vietnam. Species identification was done using PCR, MALDI-TOF and whole genome sequence (WGS). The MICs of commonly used antimicrobials was established. Thirty presumed A. hydrophila isolates were sequenced for species confirmation and genomic comparison. A phylogenetic analysis was conducted using publicly available sequences and sequences from this study. A total of 25/30 isolates were A. dhakensis sequence type (ST) 656 and 5/30 isolates were A. hydrophila ST 251. Our isolates and all publicly available A. hydrophila isolates from Vietnam belonged to ST 251 and differed with <200 single nucleotide polymorphisms (SNP). Similarly, all A. dhakensis isolates from Vietnam belonged to ST 656 and differed with <100 SNPs. The tet(A) gene was found in 1/5 A. hydrophila and 19/25 A. dhakensis. All A. hydrophila had an MIC ≤2 mg/L while 19/25 A. dhakensis had MIC ≥8 mg/L for oxytetracycline. The floR gene was only found in A. dhakensis (14/25) which showed a MIC ≥8 mg/L for florfenicol. Key virulence genes, i.e., aerA/act, ahh1 and hlyA were present in all genomes, while ast was only present in A. dhakensis. This study confirms previous findings where A. dhakensis was the dominating pathogen causing MAS and that the importance of A. hydrophila has likely been overestimated. The differences in antimicrobial susceptibility between the two species could indicate a need for targeted antimicrobial treatment plans. The lipopolysaccharide regions and outer membrane proteins did not significantly differ in their immunogenic potentials, but it remains to be determined with in vivo experiments whether there is a difference in the efficacy of available vaccines against A. hydrophila and A. dhakensis.

Retrospective Analysis of Sensitivity Characteristics of Enterobacteriaceae: A Study Based on Specimen Types, Sex, and Age Bracket of Patients.

Enterobacteriaceae have displayed widespread trends of antimicrobial resistance in recent years. Therefore, we aimed to analyse the antimicrobial susceptibility of common bacteria and explore the significance in treatment and research of infections induced by Enterobacteriaceae. We retrospectively analysed 10,775 antimicrobial susceptibility test results acquired over a 6-year period in the affiliated hospital of Chengde Medical University. We divided the data based on specimen type (blood, sputum, pus, or urine), and population characteristics (age bracket and sex) for analysis. We mainly analysed the antimicrobial susceptibility of Escherichia coli (Eco), Klebsiella pneumoniae (Kpn), and Enterobacter cloacae (Ecl). In our study, it was found that the resistance rates of Eco, Kpn, and Ecl to most antimicrobial agents were significantly different (P < 0.05) on specimen type and age bracket. The Eco from sputum had the highest resistance rates except ciprofloxacin (CIP), levofloxacin (LVX), and gentamicin (GEN); the Kpn from urine had the highest resistance rates to all antimicrobial agents; the Ecl from urine had the highest resistance rates to most antimicrobial agents. The Eco from geriatric patients had the highest resistance rates except GEN and SXT; the Kpn from adult patients had the lowest resistance rates to most antimicrobial agents except LVX. The Eco isolated from males had higher resistance rates to most antimicrobial agents except CIP, LVX, and NIT than those isolated from females; the Kpn showed significant differences in antimicrobial susceptibility to only 5 out of 22 antimicrobial agents (P < 0.05); the Ecl showed significant differences in susceptibility only to two antimicrobial agents, LVX and TOB (P < 0.01). The antimicrobial susceptibility of Enterobacteriaceae was significantly different among specimen type, age bracket and sex of patients, which is of great significance for the treatment and research of infection.

Antimicrobial Resistance and Molecular Characterization of Staphylococcus aureus Recovered from Cows with Clinical Mastitis in Dairy Herds from Southeastern Brazil.

Staphylococcus aureus is a contagious pathogen frequently associated with bovine mastitis in Brazil. Molecular characterization of Staph. aureus isolated from affected mammary quarters of cows with clinical mastitis (CM) can provide data on epidemiological behavior of this pathogen and antimicrobial susceptibility (AMS) assessment at the genotypic level. This study genotypically characterized Staph. aureus isolates recovered from cows with CM and determined the association of genotypes and AMS. A total of 84 Staph. aureus strains identified from affected mammary quarters of cows with CM in 13 dairy herds from Southeastern Brazil were submitted for susceptibility testing to 10 antimicrobials using the technique of minimal inhibitory concentration. The same isolates were also genotyped using the spa-typing methodology. Results showed a high genotypic similarity between the Staph. aureus isolates within and between herds, which were categorized as resistant to most antimicrobials, especially to β-lactam antibiotics. In addition, differences in AMS were observed among genotypic clusters, which may affect the efficacy of antimicrobials used to treat CM in different dairy herds.

Contribution of the efflux pump AcrAB-TolC to the tolerance of chlorhexidine and other biocides in Klebsiella spp.

Introduction. We are becoming increasingly reliant on the effectiveness of biocides to combat the spread of Gram-negative multi-drug-resistant (MDR) pathogens, including Klebsiella pneumoniae . It has been shown that chlorhexidine exposure can lead to mutations in the efflux pump repressor regulators SmvR and RamR, but the contribution of each individual efflux pump to biocide tolerance is unknown. Hypothesis. Multiple efflux pumps, including SmvA and AcrAB-TolC, are involved in increased tolerance to biocides. However, strains with upregulated AcrAB-TolC caused by biocide exposure are more problematic due to their increased MDR phenotype. Aim. To investigate the role of AcrAB-TolC in the tolerance to several biocides, including chlorhexidine, and the potential threat of cross-resistance to antibiotics through increased expression of this efflux pump. Methodology. Antimicrobial susceptibility testing was performed on K. pneumoniae isolates with ramR mutations selected for after exposure to chlorhexidine, as well as transposon mutants in components and regulators of AcrAB-TolC. RTPCR was used to detect the expression levels of this pump after biocide exposure. Strains from the globally important ST258 clade were compared for genetic differences in acrAB-TolC and its regulators and for phenotypic differences in antimicrobial susceptibility. Results. Cross-resistance to antimicrobials was observed following mutations in ramR. Exposure to chlorhexidine led to increased expression of acrA and its activator ramA, and transposon mutants in AcrAB-TolC have increased susceptibility to several biocides, including chlorhexidine. Variations in ramR within the ST258 clade led to an increase in tolerance to certain biocides, although this was strain dependent. One strain, MKP103, that had increased levels of biocide tolerance showed a unique mutation in ramR that was reflected in enhanced expression of acrA and ramA. MKP103 transposon variants were able to further enhance their tolerance to specific biocides with mutations affecting SmvA. Conclusions. Biocide tolerance in K. pneumoniae is dependent upon several components, with increased efflux through AcrAB-TolC being an important one.

Canine Cystitis - Biofilm Formation by Bacterial Isolates

Background: Biofilms have been reported as important virulent markers associated with drug resistance in urinary tract infections (UTIs) in humans and dogs. However, in veterinary medicine, researches involving biofilm formation, treatments and preventions have been limited; yet, it is still possible to find few studies demonstrating biofilm-forming bacteria associated with different comorbidities such as otitis, wound infections, UTIs, and endometritis. These studies generally select dogs with chronic and recurrent infections, which could be an important factor in antibiotic resistance. We aimed to evaluate biofilms in sporadic cystitis regarding prevalence and drug resistance.Materials, Methods &amp; Results: Urine samples were collected by cystocentesis from 36 client-owned dogs under clinical and laboratory suspicion of non-recurrent urinary bladder infection (cystitis). Urine was aseptically plated onto blood agar, MacConkey, and CLED, followed by incubation for 24 to 48 h. Definitive identification of a potential pathogen was made by subculture collected from an isolated colony to obtain a pure culture. The gram staining method and specific biochemical tests (phenol red fermentation, lysine, phenylalanine, citrate, sulfide-indole-motility, and urease) were used to distinguish and classify the bacteria. After identification, the bacteria were tested for antimicrobial susceptibility by a standard disk diffusion method, using the following antimicrobials: amoxicillin with clavulanic acid, ampicillin, ceftriaxone, ciprofloxacin, clindamycin, cefazolin, cephalothin, erythromycin, gentamicin, norfloxacin, and sulfamethoxazole-trimethoprim. The biofilm-forming ability was determined based on a culture in Congo red agar (CRA), where biofilm producer strains formed black colonies with a dry crystalline surface, while non-biofilm producer strains formed red colonies with a smooth surface. A crystal violet dye assay was used to confirm the CRA results. Of the 36 urine samples collected from dogs with suspected cystitis, a total of 37 isolates were obtained, from mixed or pure cultures. The most prevalent bacteria were Escherichia coli (11/37), followed by Staphylococcus spp. (8/37), Proteus spp. (7/37), and Enterococcus spp. (5/37). Other less prevalent bacteria were Klebsiella spp., Streptococcus spp., and Enterobacter spp. As for biofilm-forming ability, 67.6% (25/37) of the 37 bacterial isolates had biofilm formation in CRA and 54.05% (20/37) on the microplates containing crystal violet dye. There was no statistical difference in antimicrobial susceptibility between biofilm producer and non-biofilm producer bacteria.Discussion: We found a high proportion (&gt; 54%) of in vitro biofilm-forming ability by different bacteria, which may indicate that biofilms may also be formed in vivo, in simple cystitis. Antimicrobial resistance was not noticed in bacteria capable of forming a biofilm; however, in a future study it is important to evaluate bacterial resistance in vivo, considering the possibility of having a different response than in vitro. In addition, the problem of the presence of a biofilm in vivo is that it can nullify the antimicrobial efficacy of therapeutic agents even with in vitro susceptibility. Besides the possibility of slow or incomplete diffusion of antibiotics through the extracellular matrix of the biofilm, aspects like hydration level, pCO2, pO2, pH, pyrimidine, and divalent cation concentration that negatively influence antimicrobial activity in vitro can also cause undesirable effects at the profound layers of the biofilm. In conclusion, all of the genera of bacteria isolated from dog’s sporadic cystitis in this study were able to form a biofilm in vitro. The pathogenicity and antibiotic resistance of bacteria appears unrelated to biofilm formation in vitro.Keywords: sessile bacteria, urine, simple cystitis, antibiotic resistance.

Different breakpoints interpretation yielded distinct resistance rates to moxifloxacin of clinically significant anaerobic bacteria

The aim of this study was to describe the differences in antimicrobial susceptibility to moxifloxacin between European Committee on Antimicrobial Susceptibility Testing (EUCAST) and Clinical and Laboratory Standards Institute (CLSI) in anaerobic microorganisms. Overall, resistance to moxifloxacin appears to be high in almost all groups of anaerobes, but enormous differences in susceptibility rates between these two committees could be observed.

Application of a solid-phase microextraction-gas chromatography-mass spectrometry/metal oxide sensor system for detection of antibiotic susceptibility in urinary tract infection-causing Escherichia coli – A proof of principle study

PurposeAntibiotic resistance is widespread throughout the world and represents a serious health concern. There is an urgent need for the development of novel tools for rapidly distinguishing antibiotic resistant bacteria from susceptible strains. Previous work has demonstrated that differences in antimicrobial susceptibility can be reflected in differences in the profile of volatile organic compounds (VOCs) produced by dissimilar strains. The aim of this study was to investigate the effect of the presence of cephalosporin antibiotics on the VOC profile of extended spectrum beta-lactamase (ESBL) and non-ESBL producing strains of Escherichia coli. Material and methodsIn this study, VOCs from strains of Escherichia coli positive and negative for the most commonly encountered ESBL, CTX-M in the presence of cephalosporin antibiotics were assessed using solid-phase microextraction (SPME) coupled with a combined gas chromatography-mass spectrometry/metal oxide sensor (GC-MS/MOS) system. ResultsOur proof-of-concept study allowed for distinguishing CTX-M positive and negative bacteria within 2 ​h after the addition of antibiotics. One MOS signal (RT: 22.6) showed a statistically significant three-way interaction (p ​= ​0.033) in addition to significant two-way interactions for culture and additive (p ​= ​0.046) plus time and additive (p ​= ​0.020). There were also significant effects observed for time (p ​= ​0.009), culture (p ​= ​0.030) and additive (p ​= ​0.028). No effects were observed in the MS data. ConclusionsThe results of our study showed the potential of VOC analysis using SPME combined with a GC-MS/MOS system for the early detection of CTX-M-producing, antibiotic-resistant E. coli, responsible for urinary tract infections (UTIs).

UK Bacteroides species surveillance survey: Change in antimicrobial resistance over 16 years (2000–2016)

ObjectivesTo assess the differences in antimicrobial susceptibility of UK Bacteroides species across two distinct cohorts from 2000 to 2016. MethodsStrain identification was performed using matrix-assisted laser-desorption ionisation time of flight mass spectrometry (MALDI-TOF MS) or by partial 16S rRNA sequencing. Minimum inhibitory concentrations (MICs) were determined using agar dilution, following CLSI guidelines (CLSI, 2012; 2017). Results224 isolates were included from 2000 to 168 from 2016. Bacteroides fragilis was the most common species, comprising 68% of the 2000 cohort, and 77% in 2016. For all antimicrobials tested, there was an overall increase in the rates of non-susceptible isolates between the cohorts. ConclusionsThe antibiogram of Bacteroides species in the UK is no longer predictable. Multi-drug resistant isolates although rare, are on the rise, and require testing to guide therapy. The monitoring and surveillance of resistance trends is imperative, as is the development of standardised, robust and accessible antimicrobial susceptibility testing methodology for clinical laboratories.

Bacterial Exposure to Nickel: Influence on Adhesion and Biofilm Formation on Orthodontic Archwires and Sensitivity to Antimicrobial Agents.

The presence of nickel could modify bacterial behavior and susceptibility to antimicrobial agents. Adhesion and biofilm formation on orthodontic archwires can be a source of bacterial colonization and possible health hazards. Staphylococcus aureus was subjected to exposure and adaptation to various sub-inhibitory concentrations of nickel. Five strains of bacteria adapted to nickel in concentrations of 62.5–1000 μg/mL were tested for adhesion and biofilm formation on nickel-titanium archwires. Archwires were previously incubated in artificial saliva. Bacteria were incubated with orthodontic wire with stirring for 4 h (adhesion) and 24 h (biofilm formation). The number of adherent bacteria was determined after sonication and cultivation on the Muller-Hinton agar. Disk diffusion method was performed on all bacteria to assess the differences in antimicrobial susceptibility. Bacteria adapted to lower concentrations of nickel adhered better to nickel-titanium than strains adapted to higher concentrations of nickel (p < 0.05). Biofilm formation was highest in strains adapted to 250 and 500 μg/mL of nickel (p < 0.05). The highest biofilm biomass was measured for strains adapted to 250 μg/mL, followed by those adapted to 1000 μg/mL. Bacteria adapted to lower concentrations of nickel demonstrated lower inhibition zone diameters in the disk diffusion method (p < 0.05), indicating increased antimicrobial resistance. In conclusion, bacteria adapted to 250 μg/mL of nickel ions adhered better, demonstrated higher biofilm formation and often had higher antimicrobial resistance than other adapted and non-adapted strains.

From the Urinary Catheter to the Prevalence of Three Classes of Integrons, β-Lactamase Genes, and Differences in Antimicrobial Susceptibility of Proteus mirabilis and Clonal Relatedness with Rep-PCR.

Introduction Proteus mirabilis is a biofilm-forming agent that quickly settles on the urinary catheters and causing catheter-associated urinary tract infections. Thus, the spread of multidrug-resistant P. mirabilis isolates, with the ability to form a biofilm that carries integron, extended-spectrum β-lactamases (ESBLs), and plasmid-mediated colistin resistance genes (mcr), represents a severe threat to managing nosocomial infectious diseases. This study is aimed at surveying the prevalence of ESBL, integrase, and mcr genes of P. mirabilis, isolated from the catheter, to assess the differences in their antimicrobial susceptibility and clonal dissemination. Method Microtiter plate assay was adopted to measure biofilm formation. The antimicrobial susceptibility was assessed by the disk diffusion method. Antimicrobial resistance genes (intI1, intI2, intI3, blaTEM, blaCTX-M, blaSHV, mcr1, and mcr2) were detected by PCR. All of the isolates were characterized by repetitive sequence-based PCR. Result From 385 collected catheters in patients admitted to the intensive care unit (ICU), 40 P. mirabilis were isolated. All of the isolates could form a biofilm. Proteus spp. had intrinsic resistance to tetracycline (95%) and nitrofurantoin (92.5%), which explains the high resistance prevalence. The most widely resistant antibiotic was trimethoprim-sulfamethoxazole (75%). Thirty-three (82.5%) isolates were classified as multidrug resistance (MDR). The prevalence of intI1 and intI2 genes was 60% and 25%, respectively. In 6 (15%) isolates, both genes were detected. The most frequent ESBL gene detected in all of the isolates was blaTEM. Also, no detection for mcr1 and mcr2 antibiotic resistance genes was reported. Rep-PCR identified 39(GTG)5 types (G1–G39) of 40 isolates that 38 isolates had unique patterns. Conclusion In this study, 82.5% of isolates were MDR with high antibiotic resistance to trimethoprim-sulfamethoxazole. The intI1 and blaTEM were the most prevalent genes in the integrase and ESBL gene family. High diversity was seen in the isolates with Rep-PCR. The increasing rate of MDR isolates with a high prevalence of resistance genes could be alarming and demonstrate the need for hygienic procedures to prevent the increased antibiotic resistance rate in the future.

Comparison of antimicrobial susceptibility interpretation among Enterobacteriaceae using CLSI and EUCAST breakpoints

PurposeTo determine the difference in antimicrobial susceptibility of various antibiotics using the CLSI & EUCAST breakpoints. MethodsIn this non interventional, retrospective observational study, we reviewed minimum inhibitory concentrations (MIC) of various antibiotics routinely reported for Enterobacteriaceae clinical isolates, from an automated microbiology identification system (VITEK-2). These MICs were then analysed using both CLSI 2019 and EUCAST 2019 guidelines and classified as per the breakpoints into various categories. ResultsThe concordance rates of the antimicrobial susceptibility for various drugs ranged from 78.2% to 100% among two breakpoints. Perfect agreement with κ = 1 (p < 0.001) was observed for only three antimicrobials ceftriaxone, levofloxacin and trimethoprim-sulfamethoxazole. The changes in antimicrobial susceptibility interpretation for cefepime, ciprofloxacin, amoxicillin clavulanic acid was majorly in Intermediate category. ConclusionThe change in interpretation of the susceptibility will lead to change in the usage of antibiotics especially due to recent change in definition of I by EUCAST. There is need of more studies in this aspect to ascertain clinical implication of change in antimicrobial susceptibility.

The Identification of Acinetobacter baumannii Blood Isolates by MALDI-TOF MS, ARDRA and blaOXA-51-like Gene-Specific Real-Time PCR

The Acinetobacter calcoaceticus-Acinetobacter baumannii (Acb) complex consists of phenotypically very similar nosocomial species; A.baumannii, Acinetobacter nosocomialis, Acinetobacter pittii, Acinetobacter seifertii and Acinetobacter djikshoorniae and one environmental species A.calcoaceticus. The rapid and accurate identification of the members of Acb complex is critical as these nosocomial pathogens can show differences in antimicrobial susceptibility and clinical outcomes. The conventional phenotypic methods are slow, unreliable and less efficient for the differentiation of Acb complex species, including the A.baumannii species within the Acb complex. Although various molecular methods are available, such as amplified ribosomal DNA restriction analysis (ARDRA) and blaOXA-51-like gene specific PCR, they are usually inconvenient for the routine diagnostic laboratories. Recently, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) offers an opportunity for rapid, cost-effective and convenient bacterial identification in routine diagnostic procedures conducted in clinical laboratory. In this study, we aimed to evaluate the diagnosis performance of MALDI-TOF MS system to identify blood isolates of A.baumannii. A total of 180 nonduplicate carbapenem resistant Acb complex (strain numbers; TR1-TR60) and A.baumannii (TR61-TR180) blood isolates were collected from the intensive care units of the three university hospitals in Turkey from January 2016 to December 2016. All isolates were evaluated by using blaOXA-51-like gene specific real time (Rt-PCR) analysis, ARDRA (restriction enzymes-AluI, CfoI, MboI, MspI, RsaI) method and MALDI-TOF MS (VITEK® MS, bioMérieux, France) system. All the strains except TR10, TR31, TR35 and TR52 were identified as A.baumannii by ARDRA method. Out of 177 of all the isolates, presence of blaOXA-51-like gene was found except for TR10, TR31 and TR52 isolates. However, TR31 without the presence of blaOXA-51-like gene was identified as A.pittii using the ARDRA. Totally 176 isolates which were identified as A.baumannii by both of the methods, ARDRA and Rt-PCR- blaOXA-51-like, were accepted as a reference for the evaluation of the diagnosis performance capacity of the MALDI-TOF MS. Overall, for all 176 isolates tested, the sensitivity obtained with the MALDI-TOF MS were 99.4% with 75% specificity. The accuracy value of the method was determined as 98.9% for the identification of A.baumannii to the species level. MALDI-TOF MS is increasingly used in diagnostic microbiology for the routine identification of bacteria to the genus, species or subspecies level with high rates of sensitivity and specificity. In future, by expanding the database, MALDI-TOF MS system would possibly become the ideal method for routine diagnostic laboratories that could potentially identify more species and even determine some characteristics of antimicrobial resistance and virulence determinants.

Characterization of AmpC-hyperproducing Escherichia coli from humans and dairy farms collected in parallel in the same geographical region.

To characterize putative AmpC-hyperproducing third-generation cephalosporin-resistant E. coli from dairy farms and their phylogenetic relationships; to identify risk factors for their presence; and to assess evidence for their zoonotic transmission into the local human population. Proteomics was used to explain differences in antimicrobial susceptibility. WGS allowed phylogenetic analysis. Multilevel, multivariable logistic regression modelling was used to identify risk factors. Increased use of amoxicillin/clavulanate was associated with an increased risk of finding AmpC hyperproducers on farms. Expansion of cephalosporin resistance in AmpC hyperproducers was seen in farm isolates with marR mutations (conferring cefoperazone resistance) or when AmpC was mutated (conferring fourth-generation cephalosporin and cefoperazone resistance). Phylogenetic analysis confirmed the dominance of ST88 amongst farm AmpC hyperproducers but there was no evidence for acquisition of farm isolates by members of the local human population. Clear evidence was found for recent farm-to-farm transmission of AmpC-hyperproducing E. coli and of adaptive mutations to expand resistance. Whilst there was no evidence of isolates entering the local human population, efforts to reduce third-generation cephalosporin resistance on dairy farms must address the high prevalence of AmpC hyperproducers. The finding that amoxicillin/clavulanate use was associated with an increased risk of finding AmpC hyperproducers is important because this is not currently categorized as a highest-priority critically important antimicrobial and so is not currently targeted for specific usage restrictions in the UK.

Genotyping and antimicrobial resistance of Streptococcus uberis isolated from bovine clinical mastitis.

A genotypic characterization of Streptococcus uberis isolated from clinical mastitis (CM) in dairy cows, and the association of Strep. uberis genotypes and antimicrobial susceptibility (AMS) was performed. A total of 89 isolates identified as Strep. uberis from 86 dairy cows with CM in 17 dairy herds of Southeastern Brazil were genotyped using random amplified polymorphic DNA (RAPD) analysis. After genotyping, two clusters (I and II) were created according to RAPD types. A commercial broth microdilution test was used to determine the susceptibility of Strep. uberis isolates to 8 antimicrobials (ampicillin, ceftiofur, cephalothin, erythromycin, penicillin, penicillin+novobiocin, pirlimycin and tetracycline). For each antimicrobial, we determined the minimal inhibitory concentrations that inhibit 50% (MIC50) and 90% (MIC90) of Strep. uberis strains. Differences in AMS among genotypic clusters were evaluated using mixed regression models. Overall, a great polymorphism (56 RAPD-types) was found among Strep. uberis isolates, although a higher genetic similarity (based on the PCR bands features) was observed within herds after genotypic clustering. No differences in AMS were observed among clusters. Strep. uberis isolated from bovine CM were resistant to most antimicrobials, with the exception of cephalothin and penicillin+novobiocin.

Impact of Early Exposure to Cefuroxime on the Composition of the Gut Microbiota in Infants Following Cesarean Delivery

To assess in mothers giving birth by cesarean delivery if prophylactic antibiotics administered either before skin incision or immediately after cutting the umbilical cord influences gut microbiota colonization and antibiotic susceptibility of the gut bacteria in the newborn. Forty-two pregnant women scheduled for elective cesarean delivery were recruited at Odense University Hospital, Denmark, and randomly assigned to receive cefuroxime either before skin incision or immediately after the umbilical cord was cut. Fecal samples were collected from all infants at age 10days and 9months. Composition of the gut microbiota was determined by 16S ribosomal RNA gene amplicon high-throughput sequencing. Gram-positive cocci and Enterobacteriaceae were isolated and identified before antimicrobial susceptibility tests were performed by disk diffusion. No clear difference in the composition of the gut microbiota was observed between infants whose mothers received cefuroxime before or after cesarean delivery at neither time point, though surprisingly at 9months of age, but not at 10days of age, the number of observed species was higher in infants where mothers received cefuroxime after cord clamping. No differences in antimicrobial susceptibility of Enterobacteriaceae, Enterococcus spp, and Staphylococcus spp were seen at 10days. Timing of cefuroxime administration to mothers undergoing cesarean delivery does not have a major effect on the gut microbiota and bacterial antibiotic resistance traits in infants. Clinicaltrials.gov: NCT02072798.

Antimicrobial susceptibility patterns of Escherichia coli phylogenetic groups isolated from bovine clinical mastitis

Determination of antimicrobial susceptibility (AMS) of Escherichia coli causing clinical mastitis (CM) according to the phylogenetic groups and its association with descriptors at the cow and herd level may help improve specific strategies for treatment and control of this pathogen in dairy herds. The aims of the present study were to (a) determine the frequency of phylogenetic groups of E. coli isolated from CM in dairy cows, and its association with cow-level descriptors (parity, lactation stage, CM severity, and affected quarter position), housing system, and season; and (b) determine and compare AMS among E. coli phylogenetic groups. A quadruplex PCR method was used to classify E. coli isolates into 1 of the 7 phylogenetic groups. Minimal inhibitory concentrations were determined for 10 antimicrobials, and survival analysis was performed to evaluate the AMS differences among E. coli phylogroups. Most E. coli isolates belonged to phylogroups A (52%) and B1 (38%). None of the cow- and herd-level descriptors were associated with the E. coli phylogenetic groups. Overall, E. coli isolates were mostly susceptible to ceftiofur (96.8%), sulfadimethoxine (75.5%), and cephalothin (74.5%). Based on the survival analysis, differences in AMS between phylogenetic groups of E. coli was observed only for cephalothin, in which strains of phylogroup A were inhibited at lower minimum inhibitory concentration than strains of phylogroup B1. Results of this study indicated low susceptibility of E. coli isolates identified from CM to most antimicrobials. In addition, differences in AMS can occur among E. coli phylogenetic groups, although they may be uncommon as they were limited to only one antimicrobial (i.e., cephalothin).

Recurrence of urinary tract infections and development of urinary-specific antibiogram for kidney transplant recipients.

Urinary tract infection (UTI) recurrence and antimicrobial resistance remain a common problem in kidney transplant recipients. Whilst the use of annual institutional antibiograms may help guide appropriate empirical antibiotic selection, these non-disease specific antibiograms do not always account for patient-specific risk factors or disease-specific resistance patterns. This study determined the frequency of UTI recurrence during the first year after kidney transplantation as well as differences in antimicrobial susceptibility between an institutional antibiogram and the disease-specific antibiogram for patients following kidney transplantation. In this study, adult patients with at least one UTI during an inpatient admission within 1 year post kidney transplantation were evaluated. A disease-specific antibiogram for UTIs in kidney transplant recipients was prepared based on culture results and was compared with the annual institutional antibiograms. Of 299 kidney transplants performed during the study period, 66 subjects meet the study inclusion criteria, of whom 47% had two or more UTIs within the first year after kidney transplant. In comparison with the institutional antibiogram, Escherichia coli isolated from urine samples from kidney transplant recipients were significantly more resistant to trimethoprim/sulfamethoxazole, ceftriaxone, cefepime, ciprofloxacin and gentamicin (P<0.0001). Multiple UTIs are common in kidney transplant recipients during the first year post-transplantation. E. coli urinary isolates were significantly more resistant to multiple antibiotic drug classes in this patient population compared with the general hospital population. Antimicrobial stewardship programmes at transplant centres should consider producing disease-specific antibiograms specifically for transplant recipients to improve empirical antibiotic selection guidance.

Distribution of antimicrobial resistance determinants, virulence-associated factors and clustered regularly interspaced palindromic repeats loci in isolates of Enterococcus faecalis from various settings and genetic lineages.

Enterococcus faecalis represents an important factor of hospital-associated infections (HAIs). The knowledge on its evolution from a commensal to an opportunistic pathogen is still limited; thus, we performed a study to characterise distribution of factors that may contribute to this adaptation. Using a collection obtained from various settings (hospitalised patients, community carriers, animals, fresh food, sewage, water), we investigated differences in antimicrobial susceptibility, distribution of antimicrobial resistance genes, virulence-associated determinants and phenotypes, and CRISPR loci in the context of the clonal relatedness of isolates. Bayesian Analysis of Population Structure revealed the presence of three major groups; two subgroups comprised almost exclusively HAI isolates, belonging to previously proposed enterococcal high-risk clonal complexes (HiRECCs) 6 and 28. Isolates of these two subgroups were significantly enriched in antimicrobial resistance genes, presumably produced a polysaccharide capsule and often carried the aggregation substance asa1; distribution of other virulence-associated genes, such as esp and cyl, formation of a biofilm and gelatinase production were more variable. Moreover, both subgroups showed a low prevalence of CRISPR-Cas 1 and 3 and presence of small CRISPR2 variants. Our study confirms the importance of HiRECCs in the population of E. faecalis and their confinement to the hospital settings.